Characterization of the Net1 Cell Cycle-dependent Regulator of the Cdc14 Phosphatase from Budding Yeast

Abstract

In the budding yeast Saccharomyces cerevisiae, the multifunctional protein Net1 is implicated in regulating the cell cycle function of the Cdc14 protein phosphatase. Genetic and cell biological data suggest that during interphase and early mitosis Net1 holds Cdc14 within the nucleolus where its activity is suppressed. Upon its transient release from Net1 at late anaphase, active Cdc14 promotes exit from mitosis by dephosphorylating targets in the nucleus and cytoplasm. In this paper we present evidence supporting the proposed role of Net1 in regulating Cdc14 and exit from mitosis. We show that the NH 2 -terminal fragment Net1(1-600) directly binds Cdc14 in vitro and is a highly specific competitive inhibitor of its activity (K i ‫؍‬ 3 nM) with five different substrates including the physiologic targets Swi5 and Sic1. An analysis of truncation mutants indicates that the Cdc14 binding site is located within a segment of Net1 containing residues 1-341. We propose that Net1 inhibits by occluding the active site of Cdc14 because it acts as a competitive inhibitor, binds to a site located within the catalytic domain (residues 1-374), binds with reduced affinity to a Cdc14 C283S mutant in which an active site Cys is replaced, and is displaced by tungstate, a transition state analog known to bind in the catalytic site of protein-tyrosine phosphatases.

Global Analysis of Cdc14 Phosphatase Reveals Diverse Roles in Mitotic Processes

“…6 Additionally, mutants in Cdc14 (cdc14-1) or in the signaling pathways leading to its release (cdc15-2) show Bnr1 localized to the bud neck and Bni1 absent from the bud neck (data not shown), consistent with a functional role for Cdc14 in formin localization. Together with the biochemical evidence that Cdc14 dephosphorylates the formins, these results are consistent with the hypothesis that Cdc14 dephosphorylation is directly responsible for formin localization.…”

“…We confirmed the absence of Cdc14-dNES at the bud neck, and Bnr1 localized to the bud neck of cdc14-dNES arrested cells (data not shown). However in our experiments, Clb2 degradation was not as efficient as 6 Y. Lu, personal communication. .…”

“…The beads were washed six times with TBT buffer and resuspended in Laemmli sample buffer. To test binding of HA 6 -tagged proteins to galactose-inducible Cdc14-FLAG and Cdc14-D253A-FLAG, yeast strains were induced with 0.3% galactose and then extracted in TBT with protease inhibitor mixture and PhosSTOP phosphatase inhibitor mixture (Roche). Extracts were incubated with M2 anti-FLAG antibody (Sigma) for 1 h and then protein G-agarose beads (Roche) for 1 h. The beads were washed six times with TBT buffer and resuspended in Laemmli sample buffer.…”

The Role of the Polo Kinase Cdc5 in Controlling Cdc14 Localization

“…In the budding yeast Saccharomyces cerevisiae, the protein phosphatase Cdc14 triggers this transition by reversing cyclin-dependent kinase (CDK) phosphorylation (reviewed in (Morgan, 1999;Bardin and Amon, 2001;McCollum and Gould, 2001). The activity of Cdc14 is controlled by Cfi1/Net1, which functions as a competitive inhibitor of this protein phosphatase (Shou et al, 1999;Visintin et al, 1999;Traverso et al, 2001). The association of Cdc14 with its inhibitor is cell cycle regulated.…”

Essential global role of CDC14 in DNA synthesis revealed by chromosome underreplication unrecognized by checkpoints in cdc14 mutants

“…While in human cells a CDC14 ortholog has been recently shown to play a key role in DNA damage response (4), studies on S. cerevisiae were mostly focused on Cdc14p roles in anaphase regulation and in the exit from mitosis. The scope of Cdc14p activity in budding yeast is believed to be limited to anaphase, because Cdc14p is sequestered in the nucleolus (5) in apparently inactive form (6) at other cell cycle stages. Therefore, while Cdc14 can potentially dephosphorylate many substrates (7,8), the most studied physiological pathways are the anaphase pathways (FEAR and MEN), which are both dependent on the two sequential bursts of Cdc14 release (1,9).…”